Heterozygous frameshift variants in HNRNPA2B1 cause early-onset oculopharyngeal muscular dystrophy

Missense variants in RNA-binding proteins (RBPs) underlie a spectrum of disease phenotypes, including amyotrophic lateral sclerosis, frontotemporal dementia, and inclusion body myopathy. Here, we present ten independent families with a severe, progressive muscular dystrophy, reminiscent of oculopharyngeal muscular dystrophy (OPMD) but of much earlier onset, caused by heterozygous frameshift variants in the RBP hnRNPA2/B1. All disease-causing frameshift mutations abolish the native stop codon and extend the reading frame, creating novel transcripts that escape nonsense-mediated decay and are translated to produce hnRNPA2/B1 protein with the same neomorphic C-terminal sequence. In contrast to previously reported disease-causing missense variants in HNRNPA2B1, these frameshift variants do not increase the propensity of hnRNPA2 protein to fibrillize. Rather, the frameshift variants have reduced affinity for the nuclear import receptor karyopherin β2, resulting in cytoplasmic accumulation of hnRNPA2 protein in cells and in animal models that recapitulate the human pathology. Thus, we expand the phenotypes associated with HNRNPA2B1 to include an early-onset form of OPMD caused by frameshift variants that alter its nucleocytoplasmic transport dynamics.

Immunostaining of patient 1 muscle biopsy with αβ-crystallin, filamin C, desmin and FHL1 showing absence of cytoplasmic or perinuclear inclusions or protein aggregates that contain these Z-disk associated myofibrillar proteins. Scale bar, 100 μm. The micrographs shown are representative images of a single diagnostic muscle biopsy in each indicated patient. No independent replicates were performed.  Supplementary Fig. 2. Frameshift HNRNPA2B1 variant transcripts are stable in muscle tissue and cause rearrangement of amino acid sequence in the C terminus. a, cDNA sequence of HNRNPA2 (NM_002137) in exon 10 and exon 11. Red nucleotides indicate nucleotides deleted in patients; underlined nucleotides indicate nucleotides deleted in patient 8. The original stop codon (TGA) is indicated in bold. b, Amino acid sequences in WT and each frameshift mutant. The consensus PY-NLS motif in M9-NLS is underlined in red on bottom row. c, RNA was extracted from muscle tissue of patients 1 (c.992delG) and 2 (c.981delA). After reverse transcriptase reaction, the cDNA was amplified by PCR and sequenced. The intensity of the chromatogram peaks corresponding to the reference and frameshift alleles appear nearly equal, suggesting little to no degradation of the frameshift transcript in muscle tissue.     showed a severe pectus excavatum with a Haller index of 13 (normal < 2.5). Brain MRI at 7 years of age was normal. Serum screen for Paget's disease of the bone was normal.
Family 2. Patient 2 is a 17-year-old young man of Northern European descent who was first noted to have difficulty with feeds in the neonatal period. His early motor development was otherwise unremarkable. Ptosis and ophthalmoplegia were noted at approximately 5 years of age.
He subsequently developed mild lower extremity predominant weakness involving the proximal muscles more than distal ones. Swallowing difficulties were also progressive and persistent.
There was no history of respiratory insufficiency. Examination at 17 years of age showed bilateral ptosis, abduction and adduction deficit of the eye movements bilaterally and mild facial weakness. Contractures were not present. Muscle strength testing showed mild proximal greater than distal weakness. He had a normal gait. Mild scoliosis was also noted. Family history was non-contributory. A de novo heterozygous variant in HNRNPA2B1 c.981delA, p.(G328Afs*31)/c.1017delA (p.G340Afs*31) was identified on whole exome sequencing. Serum CK was 602 units/L. NCS/EMG was not performed. Muscle biopsy obtained at 13 years of age from the left vastus lateralis muscle showed fiber size variability and rimmed vacuoles. Electron microscopy identified cytoplasmic, membrane-bound autophagic vacuoles and tubulofilamentous inclusions predominantly in the cytoplasm and peri-nuclear areas. A few angular atrophic fibers were also present. There was no inflammation or mitochondrial abnormalities. Brain MRI was normal.
Family 3. Patient 3 is a 17-year-old young woman of Ukrainian descent who was born premature at 33 weeks of gestation with bilateral congenital hip dysplasia. She had delays in reaching motor milestones. Ptosis and ophthalmoplegia were noted by 2.5 years of age, and global muscle weakness was apparent by 6 years of age. Her symptoms have been slowly progressive over time. Examination revealed ptosis, ophthalmoplegia, facial weakness and dysphonia. Mild proximal lower and upper extremity weakness was noted. Mild scoliosis was also present.
Reflexes were reduced and gait exam showed a hyperlordotic posture. She was unable to run.  The limb weakness was most marked proximally and necessitated wheelchair use by her late 50s.
The younger daughter, Patient 4, presented at 18 years of age with ophthalmoplegia and ptosis.
Her symptoms progressed and she also noted a poor cough and significant dysphagia, and proximal limb weakness in her 30s. Serum CK was 808 units/L. The older daughter, Patient 5, presented at 17 years of age with bilateral ptosis, ophthalmoplegia, and dysphagia. By her 40s she developed proximal upper and lower extremity weakness with progression of ophthalmoplegia and significant dysphagia. Serum CK was 1105 units/L. She also had respiratory insufficiency (FVC 45% predicted). Quadriceps muscle biopsy at 40 years of age showed significant variation in muscle fiber size with an increased number of internal nuclei and rimmed vacuoles. There was no evidence of inflammation or mitochondrial abnormalities.
Electron microscopy was reported as normal. All affected family members carried a heterozygous variant in HNRNPA2B1 c.966delA, p.(N323Tfs*36)/c.1002delA;p.(N335Tfs*36) with a dominant pattern of inheritance. OPMD gene analysis showed no evidence of an expansion of the GCG repeat sequence within PABN1. There were no abnormalities of bone biochemistry and no evidence of dementia or cognitive decline.
Family 5. Patient 6 is a 12-year-old girl of English ancestry who developed respiratory difficulties immediately after birth with subsequent tracheostomy, which remained in place at the time of evaluation. She also was noted to have a hemifacial macrosomia complex with left anophthalmia. She had mild delays in reaching gross motor milestones but was independently ambulatory with mild proximal greater than distal weakness. Her gait was waddling in quality with a hyperlordotic posture. She also had ptosis and complete ophthalmoplegia and dysphagia, requiring a percutaneous gastrostomy tube for nutrition. She had a jaw hamartoma that was resected. She also had respiratory insufficiency (FVC 21% predicted). Mild large joint contractures were present. Serum CK was 770 units/L. EMG/NCS showed an irritable myopathy with myotonic and pseudomyotonic discharges. Repetitive nerve stimulation and SFEMG was normal. Family history was non-contributory. A presumed de novo heterozygous variant in HNRNPA2B1 c.1001delG, p.(G334Vfs*25)/c.1037delG;p.(G346Vfs*25) was identified on whole exome sequencing.
Family 6. Patient 7 is a 9-year-old French girl whose gestation was notable for reduced fetal movements. She was born with intrauterine growth retardation. Ptosis was noted by her parents at birth, which became more pronounced with fatigue and as she aged. Respiratory impairment began at 6 months of age with bronchiolitis and recurrent pneumonias. She walked at 14-15 months of age and did not have a speech delay. Axial weakness and ophthalmoplegia were noted at 4.5 years of age. At age 5, she also developed dysphonia and dysphagia, requiring a percutaneous gastrostomy tube for nutrition and respiratory insufficiency necessitated the use of nocturnal non-invasive ventilatory support. At age 8, she her respiratory insufficiency worsened and she required continuous non-invasive ventilation and her muscle weakness progressed, resulting in loss of independent ambulation and use of a motorized wheelchair. Serum CKs ranged between 287 and 536 units/L. NCS/EMG was normal except for increased latency of tendon reflexes (T reflex). Repetitive nerve stimulation was normal. Quadriceps muscle biopsy obtained at 4 years and 9 months of age showed myofiber necrosis and regeneration, and subsarcolemmal vacuoles. Family history was non-contributory. A de novo heterozygous variant in HNRNPA2B1 c.996_997dup, p.(G333Vfs*27)/c.1032_1033dup; p.(G345Vfs*27) was identified on trio-based whole exome sequencing.
Family 7. Patient 8 is a 20-year-old French man with delays in achieving early motor milestones as an infant. During childhood, he had a propensity to fall and had difficulty participating in sports. He had ptosis, ophthalmoplegia and dysphonia. He also had axial and proximal greater than distal weakness in all limbs that was progressive, resulting in loss of independent ambulation. He also has respiratory insufficiency and uses non-invasive ventilatory support at nighttime. Large joint contractures and scoliosis were also noted. Serum CK was 2484 units/L. NCS/EMG was consistent with a myopathic process with fibrillation potentials at rest. Repetitive nerve stimulation was normal. Muscle biopsy at 14 years of age showed variability in muscle fiber size, myofiber atrophy, and cytoplasmic rimmed vacuoles and internalized nuclei. There was also a moderate increase in the endomysial connective tissue. Electron microscopy studies showed marked autophagic changes and vacuoles containing membranous, myelin-like remnants. Many myofibrils contained areas with tubulofilamentous inclusions, which on occasion were also present in myonuclei. Brain MRI was normal. Family history was noncontributory. Next generation sequencing panels including LGMD genes and congenital myopathy genes were non-diagnositc. Due to the similarity of the phenotype with other cases in this cohort, Sanger sequencing of the HNRNPA2B1 gene was pursued. A de novo heterozygous variant in HNRNPA2B1, c.980_986del, p.(G327Vfs*30)/c.1016_1022del, p.(Gly339Valfs*30), was identified.
Family 8. Patient 9 is a 37-year-old man born of non-consanguineous parents of Italian descent.
There was no delay in the acquisition of psychomotor milestones, although he was reported to always have been slower than his peers in running. Starting at 5 years of age, he developed progressive muscle weakness especially in the lower limbs, skeletal deformities of talipes equinovarus and scoliosis, bilateral ptosis and ophthalmoplegia. He lost the ability to run at 13 years of age and lost the ability to independently ambulate at 26 years of age. He underwent a blepharoplasty for severe bilateral ptosis at 28 years of age. He also developed a severe dysphagia and currently needs a semi-liquid diet and thickened liquids. Echocardiography was normal and pulmonary function testing showed a restrictive defect (FVC 34% predicted), resulting in the need for non-invasive nocturnal ventilation since 31 years of age. At 34 years of age, he underwent surgery for a retraction of the temporomandibular joint. The most recent neurological examination (35 years of age) showed severe weakness in all extremities with antigravity movements only possible in the distal segments of upper limbs, inability to sit without support, absent deep tendon reflexes, normal sensation, surgically corrected ptosis, complete loss of vertical and near-complete loss of horizontal eye movements, moderate facial weakness, hypomobility of the velum palatinum and a hypotrophic tongue. Serum CK was elevated between 280 and 7067 units/L. A muscle biopsy at age 9 years showed nonspecific myopathic changes, while a second biopsy at age 13 was notable for a severe myopathic picture, with corelike areas of reduced oxidative reaction in both fiber types and rimmed vacuoles. Genetic testing for OPMD (GCN repetitions in PABPN1) was normal. Next generation sequencing (Illumina Trusight One expanded panel of human disease-causing genes) identified a heterozygous c.974delG, p.(G325Vfs*34)/c.1010delG, p.(Gly337ValfsTer34) variant in HNRNPA2B1 which was confirmed to be de novo by Sanger sequencing of parental samples.
Family 9. Patient 10 is a 7-year-old girl of Indian origin who presented with a respiratory infection at 18 months of age with acute onset bilateral ptosis lasting a week. Her early motor milestones and motor function were normal until 4.5 years of age. Axial weakness, ptosis and ophthalmoplegia were first noted at 4.5 years of age, coinciding again with a respiratory illness.
She also developed dysphonia and dysphagia, requiring a percutaneous gastrostomy tube for nutrition at 6 years of age. She has a weak cough. Her weakness in the extremities developed and progressed, resulting in loss of independent ambulation by 7 years of age. She also has long finger flexor and ankle contractures. She demonstrates increased weakness during intercurrent chest infections. Serum CK levels were elevated at 424 units/L. NCS/EMG was consistent with a myopathic process. Stimulated, single fiber EMG was normal and brain MRI was unremarkable.
Plasma lactate was 3.9; urine organic acids showed strongly raised pyruvate with mildly raised lactate and respiratory chain studies detected low complex I and IV activity. Mitochondrial DNA sequencing did not reveal any pathogenic variants. Quadriceps muscle biopsy obtained at 5 years and 6 months of age showed rimmed vacuoles, sparse protein aggregates and few central/internal nuclei with scattered basophilic coarsely granular fibers, some of which contained large eosinophilic inclusions. Acid phosphatase activity was high in many of the rimmed vacuoles.
Family 10. Patient 11 is an 18-year-old woman of Japanese origin with normal motor milestones, who was noted to be a slow runner in childhood. At 10 years of age, short stature was noted; however, growth hormone levels were within the normal range. She developed progressive weakness: at 15 years of age, she was noted to have easy fatiguability and difficulty running and climbing stairs. On examination, bilateral ptosis, ophthalmoplegia, lower proximal muscle weakness, scoliosis, and waddling gate were noted at 17 years of age. She has a decreased forced vital capacity (FVC) of 58% predicted. Serum CK levels were elevated at 501 Units/L. NCS was normal. Brain MRI was unremarkable. Muscle CT revealed severely affected thigh muscle with relative sparing of the rectus femoris, gracilis, and adductor longus muscle.